ABSTRACT Interruption and subsequent restoration of circulation in tissues causes varying amounts of ischemia- reperfusion injury (IRI). IRI is a detrimental feature of many clinical events such as organ transplants, cardiopulmonary bypass (CPB) used in cardiac surgery, myocardial infarction (MI), and stroke. There is currently no effective therapy to prevent or reduce IRI. This proposal focuses on cerebral IRI, a significant clinical problem contributing to considerable morbidity and large numbers of deaths. When an artery is occluded, there is an area of tissue that is deprived of oxygen and nutrients, leading to necrosis and infarction. However, there is a large border zone around the infarction that does not get sufficient circulation to function normally, but enough to survive. This area at risk (AAR), known in the brain as the penumbra, experiences IRI, even without acute restoration of circulation. IRI is associated with early increased endothelial permeability and edema and later, changes in gene expression, resulting in inflammation. There is growing awareness of the vital role played by the innate immune system in IRI. The innate immune system is comprised of extracellular and intracellular receptors that activate multiple signaling molecules controlling gene transcription leading to an inflammatory phenotype. This very complex system evolved over 700 million years to protect uni- and multi- cellular organisms from infection with bacteria, fungi, viruses and other pathogens. Involvement of the innate immune system in IRI is puzzling - there is no infection, although there is tissue damage due to infarction that is misinterpreted by the receptors of the innate immune system as infection. This concept - that IRI is a biologic mistake - is the basis of X-In8 Biologicals Corp. Inhibiting activation of the innate immune system temporarily may have a profound impact on IRI and dramatically reduce the size of the penumbra, resulting in much less tissue damage and smaller strokes. Our research has implicated TLR4 as a mediator of lung edema and inflammation due to lung IRI. Thus, we are proposing to test CRX-526, a potent TLR4 inhibitor, for efficacy to prevent and/or reduce cerebral IRI. Our initial target applications are prevention of IRI during stroke and treatment of acute cerebral artery occlusion. However, because the innate immune system is involved in the delayed phase of IRI, therapeutic intervention soon after a stroke may be highly beneficial as well. Beyond cerebral IRI, any successful therapy to reduce IRI would have a profound impact on organ transplantation, myocardial damage due to CPB, and the natural history and treatment for MI and stroke. PUBLIC HEALTH RELEVANCE: Ischemia-reperfusion injury (IRI) is a major problem in many clinical scenarios, including organ transplantation, myocardial infarction, and stroke. When a cerebral artery is occluded due to embolic stroke, inflammation and edema due to IRI is responsible for a large amount of neuronal cell death in the penumbrum, a large zone around the infarcted area. Because toll-like receptors (TLRs) of the innate immune system, particularly TLR4 and TLR2, mediate much of the inflammation associated with IRI in many organs, including the brain, this proposal outlines novel experiments to determine the impact of inhibiting TLR4 activation with a potent TLR4 inhibitor, that appears to have some inhibitory effects on TLR2 in endothelial cells, through collaboration of a university-based expert in cerebral IRI, and X-In8, a new biotechnology company with a novel concept about IRI. In Phase I we propose to evaluate the impact of TLR4 inhibition using an established in vivo model of brain IRI, and proven methods to quantify edema due to brain IRI. When we determine a benefit of TLR4 inhibition in this model, in Phase 2 we propose to determine efficacy of this approach in translational small and large animal models of stroke and acute interventions to restore circulation to ischemic areas of brain.